Gillian E. Caughey

Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH2, the common substrate for thromboxane A2 (TXA2), prostacyclin (PGI2), and PGE2 synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1β caused induction of COX-2. TXA2 was the predominant COX-1-derived product, and TXA2 synthesis changed little with up-regulation of COX-2 by IL-1β (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI2 and PGE2 (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI2 and PGE2 synthesis, but had little effect on TXA2 synthesis. The up-regulation of COX-2 by IL-1β was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA2 synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI2 and PGE2 synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI2 and PGE2 linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.

Differential Regulation of Prostaglandin E2 and Thromboxane A2 Production in Human Monocytes: Implications for the Use of Cyclooxygenase Inhibitors

There is an autocrine relationship between eicosanoid and cytokine synthesis, with the ratio of prostaglandin E2 (PGE2)/thromboxane A2 (TXA2) being one of the determinants of the level of cytokine synthesis. In monocytes, cyclooxygenase type 1 (COX-1) activity appears to favor TXA2 production and COX-2 activity appears to favor PGE2 production. This has led to speculation regarding possible linkage of COX isozymes with PGE and TXA synthase. We have studied the kinetics of PGE2 and TXA2 synthesis under conditions that rely on COX-1 or -2 activity. With small amounts of endogenously generated prostaglandin H2 (PGH2), TXA2 synthesis was greater than PGE2. With greater amounts of endogenously generated PGH2, PGE2 synthesis was greater than TXA2. Also, TXA synthase was saturated at lower substrate concentrations than PGE synthase. This pattern was observed irrespective of whether PGH2 was produced by COX-1 or COX-2 or whether it was added directly. Furthermore, the inhibition of eicosanoid production by the action of nonsteroidal anti-inflammatory drugs or by the prevention of COX-2 induction with the p38 mitogen-activated protein kinase inhibitor SKF86002 was greater for PGE2 than for TXA2. It is proposed that different kinetics of PGE synthase and TXA synthase account for the patterns of production of these eicosanoids in monocytes under a variety of experimental conditions. These properties provide an alternative explanation to notional linkage or compartmentalization of COX-1 or -2 with the respective terminal synthases and that therapeutically induced changes in eicosanoid ratios toward predominance of TXA2 may have unwanted effects in long-term anti-inflammatory and anti-arthritic therapy.

Effects of Hypoxia on Monocyte Inflammatory Mediator Production DISSOCIATION BETWEEN CHANGES IN CYCLOOXYGENASE-2 EXPRESSION AND EICOSANOID SYNTHESIS

Blood-derived monocytes are found at sites of inflammation as well as in solid tumors and atherosclerotic arteries. They are an abundant source of inflammatory eicosanoids such as prostaglandin E2 (PGE2) and thromboxane A2, which are formed via arachidonic acid (AA) metabolism by cyclooxygenase-1/2 (COX-1/2). In vitro studies of inflammatory mediator production are conducted invariably in room air, which does not reflect the oxygen tensions found in monocyte-containing lesions, which are frequently hypoxic. In this work we examined the effects of hypoxia at levels reported in these lesions, on monocyte COX-2 expression, the related events that lead to eicosanoid synthesis, and relationships with tumor necrosis factor (TNF)-α synthesis. In fresh human monocytes exposed to hypoxia (1% O2), there was an increase in COX-2 protein compared with cells in normoxia, and this was attributable to increased transcription and mRNA stability. However, the synthesis of PGE2 and thromboxane A2 was reduced in hypoxia and did not reflect the increased level of COX-2. Monocytes prelabeled with [3H]AA followed by lipopolysaccharide stimulation in the presence of hypoxia showed a reduced release of AA compared with cells in normoxia. In addition, hypoxia resulted in decreased phosphorylation of the p44/42 mitogen-activated protein kinase and of cytosolic phospholipase A2. Hypoxia also increased TNF-α synthesis, which appeared to play a role in COX-2 expression, and the observed increase TNF-α synthesis appeared to result from reduced PGE2 synthesis. Overall, the results suggest the existence of an autocrine loop of regulation between monocyte eicosanoid and TNF-α production, which is dysregulated in hypoxia and establishes hypoxia as being an important environmental determinant of inflammatory mediator production.

Prevalence of preventable medication-related hospitalizations in Australia: an opportunity to reduce harm.

OBJECTIVE To identify the prevalence of potentially preventable medication-related hospitalizations amongst elderly Australian veterans by applying clinical indicators to administrative claims data. DESIGN AND SETTING Retrospective cohort study in the Australian veteran population from 1 January 2004 to 31 December 2008. PARTICIPANTS A total of 109 044 veterans with one or more hospitalizations defined by the medication-related clinical indicator set, during the 5-year study period. MAIN OUTCOME MEASURE The prevalence of potentially preventable medication-related hospitalizations as a proportion of all hospitalizations defined by the clinical indicator set. RESULTS During the 5-year study period, there were a total of 1 630 008 hospital admissions of which 216 527 (13.3%) were for conditions defined by the medication-related clinical indicator set for 109 044 veterans. The overall proportion of potentially preventable medication-related hospitalizations was 20.3% (n= 43 963). Of the 109 044 veterans included in the study, 28 044 (25.7%) had at least one potentially preventable medication-related hospitalization and 7245 (6.6%) veterans had two or more potentially preventable admissions. Conditions with both a high prevalence of hospitalization and preventability included asthma/chronic obstructive pulmonary disorder, depression and thromboembolic cerebrovascular event (23.3, 18.5 and 18.3%, respectively, were potentially preventable). Other hospitalizations that were less common but had a high level of preventability (at least 20%) included hip fracture, impaction, renal failure, acute confusion, bipolar disorder and hyperkalaemia. CONCLUSIONS The results of this study highlight those conditions where hospitalizations could potentially be avoided through improved medication management. Strategies to increase the awareness, identification and resolution of these medication-related problems contributing to these hospitalizations are required in Australia.

Comorbid chronic diseases, discordant impact on mortality in older people: a 14-year longitudinal population study

Objectives To determine the impact of comorbid chronic diseases on mortality in older people. Design Prospective cohort study (1992–2006). Associations between numbers of chronic diseases or mutually exclusive comorbid chronic diseases on mortality over 14 years, by Cox proportional hazards model adjusting for sociodemographic variables or Kaplan–Meier analyses, respectively. Setting Population based, Australia. Participants 2087 randomly selected participants aged ≥65 years old, living in the community or institutions. Main results Participants with 3–4 or ≥5 diseases had a 25% (95% CI 1.05 to 1.5, p=0.01) and 80% (95% CI 1.5 to 2.2, p<0.0001) increased risk of mortality, respectively, by comparison with no chronic disease, after adjusting for age, sex and residential status. When cardiovascular disease (CVD), mental health problem or diabetes were comorbid with arthritis, there was a trend towards increased survival (range 8.2–9.5 years) by comparison with CVD, mental health problem or diabetes alone (survival 5.8–6.9 years). This increase in survival with arthritis as a comorbidity was negated when CVD and mental health problems or CVD and diabetes were present in disease combinations together. Conclusion Older people with ≥3 chronic diseases have increased risk of mortality, but discordant effects on survival depend on specific disease combinations. These results raise the hypothesis that patients who have an increased likelihood of opportunity for care from their physician are more likely to have comorbid diseases detected and managed.

Major bleeding risk associated with warfarin and co‐medications in the elderly population

Warfarin management in the elderly population is complex as medicines prescribed for concomitant diseases may further increase the risk of major bleeding associated with warfarin use. We aimed to quantify the excess risk of bleeding‐related hospitalisation when warfarin was co‐dispensed with potentially interacting medicines.

Stroke risk and NSAIDs: an Australian population-based study

Objective: To determine the risk of stroke associated with non‐steroidal anti‐inflammatory drug (NSAID) use.

Eicosanoid production by human monocytes: does COX-2 contribute to a self-limiting inflammatory response?

Abstract: The eicosanoids, prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), are involved in inflammatory events. TXA2 has potentially pro-inflammatory actions and PGE2 has actions which can be considered both pro- and anti-inflammatory. Therefore, it is potentially significant that production of TXA2 and PGE2 by stimulated monocytes have very different time courses. TXA2 synthesis is immediate and dependent on cyclooxygenase Type 1 (COX-1) activity whereas PGE2 synthesis is delayed and dependent on COX-2 activity. These apparent COX-isotype dependencies of TXA2 and PGE2 synthesis can be explained by differences in the affinities of TXA synthase and PGE synthase for the common substrate, PGH2. The findings have implications for the use of NSAIDs and selective COX-2 inhibitors whose actions can increase the monocyte TXA2/PGE2 ratio.

Assay of cyclooxygenase-1 and 2 in human monocytes

Abstract.Objective and Design: There is frequently poor correlation between in vitro methods for calculated cyclooxygenase (COX)-1/COX-2 selectivities of inflammatory agents. Therefore, we have examined the use of a single stimulus in a single cell type containing both COX isoforms, for determining the selectivities of COX-inhibitory agents.¶Methods: Fresh human monocytes were stimulated with arachidonic acid (AA; 10 μM) for 15min and prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production were used as a measure of COX-1 activity. To measure COX-2 activity, cells were transiently pre-treated with aspirin to irreversibly inhibit constitutive COX-1, treated with lipopolysaccharide (LPS) to induce COX-2 and then stimulated with AA.¶Results: Eicosanoid production in resting monocytes was predominantly COX-1 derived since it was not inhibited by NS-398 and also, COX-2 was not detectable. In LPS treated monocytes pre-treated transiently with aspirin, neither the level of induced COX-2 nor the activity was affected. Using the mean of the results for PGE2 and TXB2 inhibition, the COX-1/COX-2 ratios of the IC50 values for aspirin and NS-398 are <0.1 and >130, respectively.¶Conclusions: This study has provided a system for investigating inhibition of COX isotypes without the potentially confounding effects of using different cell types with different stimuli for each isotype as seen in other published systems. Dose responses to aspirin and NS-398 which are COX-1 and COX-2 selective inhibitors respectively, confirmed the utility of this system.

Comorbid Diabetes and COPD: Impact of corticosteroid use on diabetes complications

OBJECTIVE To identify if there is a dose-dependent risk of diabetes complications in patients treated with corticosteroids who have both diabetes and chronic obstructive pulmonary disorder (COPD). RESEARCH DESIGN AND METHODS A retrospective study of administrative claims data from the Australian Government Department of Veterans’ Affairs, from 1 July 2001 to 30 June 2008, of diabetes patients newly initiated on metformin or sulfonylurea. COPD was identified by dispensings of tiotropium or ipratropium in the 6 months preceding study entry. Total corticosteroid use (inhaled and systemic) in the 12 months after study entry was determined. The outcome was time to hospitalization for a diabetes-related complication. Competing risks and Cox proportional hazard regression analyses were conducted with adjustment for a number of covariates. RESULTS A total of 18,226 subjects with diabetes were identified, of which 5.9% had COPD. Of those with COPD, 67.2% were dispensed corticosteroids in the 12 months from study entry. Stratification by dose of corticosteroids demonstrated a 94% increased likelihood of hospitalization for a diabetes complication for those who received a total defined daily dose (DDD) of corticosteroids ≥0.83/day (subhazard ratio 1.94 [95% CI 1.14–3.28], P = 0.014), by comparison with those who did not receive a corticosteroid. Lower doses of corticosteroid (<0.83 DDD/day) were not associated with an increased risk of diabetes-related hospitalization. CONCLUSIONS In patients with diabetes and COPD, an increased risk of diabetes-related hospitalizations was only evident with use of high doses of corticosteroids. This highlights the need for constant revision of corticosteroid dose in those with diabetes and COPD, to ensure that the minimally effective dose is used, together with review of appropriate response to therapy.